Lyophilization of mRNA-LNP solution

Background

Cryogenic preservation and transportation are needed for the two current licenced mRNA vaccines (<-60°C). The stringent requirements come from the complex interactions among multiple lipid components and the instability of mRNA, which is sensitive to oxygen, moisture, enzymes, pH, and so on.

Lyophilization, a process that removes water by sublimation under vacuum at a low temperature, seems to be a promising solution. It is a relatively mild drying method applicable to vulnerable biomacromolecules or colloidal nanoparticles.

Problems

Lyophilization of mRNA-LNP is more complex than of small molecules or proteins. Conventional freeze-drying process tends to cause aggregation and rupture of nanoparticles.

Techniques

Enobio optimized the lyophilization process from multiple factors, such as lyophilization pressure and temperature, buffer and lyoprotectant.

Result

Enobio has repeatedly screened and optimized for every detail, and systematically summarized the experience of mRNA-LNP lyophilization, which led to excellent lyophilization result for different mRNA and lipid components.

Lyophilized mRNA-LNP samples

Lyophilization with different mRNAs

Lyophilization with different LNPs

No significant changes after lyophilization

Invivo activity of the lyophilized formulation was not affected

Comparison of Luciferase mRNA-LNP expression efficiency in vivo before and after lyophilization, Balb/c mice were injected with 5 μg of mRNA-Luc LNP in the tail vein, and imaged 24 h later, which showed that there was no significant change in fluorescence brightness before and after lyophilization.

Thevaccine still induces the same immune response after lyophilization

Balb/c mice were immunized with 5 μg of mRNA-WT LNP before and after lyophilization of D0/D7, and blood was collected on D21 to detect the titers of binding antibody and pseudovirus-neutralizing antibodies, which showed that there was no difference in immunogenicity before and after lyophilization.

No new impurities were generated by HPLC-CAD analysis of lyophilized mRNA-LNP after 6 months at 25℃.

The rate of hydrolytic oxidation of lipids and mRNA decreases dramatically in lyophilized forms

No new impurities were generated by HPLC-CAD analysis of lyophilized mRNA-LNP after 6 months at 25℃.

Excellent stability of lyophilized samples

Comparison of the stability of lyophilized formulations under different storage conditions.

Applications in COVID-19 vaccine

Lyophilized vaccine elicit significant immune responses

(a-b)：C57BL/6NmiceD0/D21immunized with different doses of lyophilized Omicron mRNA-LNP, and collecting blood to detect that t i ters of binding antibody and neutralizing antibody

Lyophilized vaccine also provides good immunization in New Zealand rabbits and aged rhesus monkeys

(c)：New Zealand rabbit immunized with lyophilized vaccine, D35 testing for neutralizing antibody levels against different Omicron strains.

(d)：Rhesus monkey D0/21 immunized with lyophilized vaccine, and the change of neutralizing antibody level against different Omicron strains was detected.

Clinical Study - Freeze-Dried Vaccine Effective Against Different Strains as Booster Shot

(a-c) : On the basis of two doses of nactivated vaccine the subiects had been 6 months from the ast dosel. the third dose of RH109 mRNA vaccine was given, and the third dose of inactivated vaccine as the control group.

(d) Neutralizino antibody fiters in serum acainst the diferent mutants were measured on day 14 ater immunizaton with the RH109 mRNA vaccine p Afer vaccination, none of the subjects had secondary or higher adverse reactons, and none of them had a body temperature rise of more than 37.5 ".

This vaccine is used as a booster shot for inactivaied vacines and has excelent safety and immunization effects aoainst prototvpe strains and Omicron variants.

Conclusion

ENO biotechnology presents an optimized lyophilization technique with precise temperature control and lower residual water content, which could effectively maintain the physiochemical properties and bioactivity of mRNA-LNPs during long-term storage at 2~8°C, room temperature and even 40°C. The improved thermostability was verified with mRNA-LNPs containing different mRNA molecules, demonstrating the wide applicability of the technique.